Light scattering is a non-invasive technique for characterizing macromolecules and a wide range of particles in solution. The two types of light scattering detection frequently used for the characterization of macromolecules are static light scattering (SLS) and dynamic light scattering (DLS).
Static light scattering is also known as multi-angle light scattering (MALS). SLS experiments involve the measurement of the absolute intensity of the light scattered from a sample in solution. This measurement allows the determination of the size of the sample molecules or particles, and, when coupled with knowledge of the sample concentration, allows for the determination their weight average molar mass. In addition, nonlinearity of the intensity of scattered light as a function of sample concentration may be used to measure interparticle interactions and associations.
Dynamic light scattering is also known as quasi-elastic light scattering (QELS) and photon correlation spectroscopy (PCS). In a DLS experiment, time-dependent fluctuations in the scattered light signal are measured using a fast photodetector. DLS measurements determine the diffusion coefficient of the molecules or particles, which can in turn be used to calculate their hydrodynamic radius.
Extensive literature has been published describing methods for making both static and dynamic light scattering measurements in flowing and batch (non-flowing) systems. See, for example, P. J. Wyatt, “Light scattering and the absolute characterization of macromolecules,” Analytica Chimica Acta, 272, 1-40, (1993). With the development and improvement in the optical quality of multiwell plates, it has become possible to make both SLS and DLS measurements directly from samples contained therein. Methods capable of measuring samples directly in these multiwell plates are generally desirable given both the high-throughput nature of the measurements and the reduced sample volume requirements. Standard multiwell plates have 96, 384, or 1536 wells, each well is able to contain a different, distinct sample, and all wells may be tested in a single data collection run. In addition, use of these plates obviates the laborious need to clean and dry individual scintillation vials or cuvettes after each measurement. These plates generally have very low volume wells, and commercially available multiwell plate based measurement instruments are capable of light scattering measurements from sample volumes of 1 μL or less. These tiny sample volumes are of great benefit when a limited amount of sample is available from which to make measurements, particularly when compared to the 300 μL or larger sized measurement volumes often required by other light scattering techniques.
Multiwell plates, however, suffer from three primary issues which can make both DLS and SLS measurements difficult to perform in the wells themselves and may produce unreliable results. These issues are: 1) high background signal originating from sidewalls and other interfaces, 2) non-uniformity of the fluid meniscus shape and level from well to well, and 3) evaporation.
The deleterious effects of high background signal, or noise, is caused by light scattered from anything other than the sample. This background signal decreases the light scattering instrument's sensitivity due to the increase in the noise present in relation to the useful signal scattered from the sample itself, and therefore an overall reduction in the signal-to-noise ratio upon which the sensitivity of the measurement is dependent. High background is primarily due to scattering from interfaces traversed by the light beam and secondary scattering of interface flare from sidewalls and other surfaces present in a multiwell scattering measurement. It is an objective of this invention to minimize the number of surfaces which the beam traverses likely to result in background scatter. For DLS measurements, higher sample concentrations of valuable sample materials are generally required to overcome this background signal. A further objective of this invention is that by the mitigation of background scatter, DLS measurements may be performed at lower concentrations than have been possible heretofore.
Non-uniformity of the fluid meniscus shape from well to well causes variability in the background signal. FIG. 1 shows a simple ray diagram in a single well for a conventional DLS measurement in a multiwell plate such as that performed by many DLS plate readers, such as the DynaPro® Plate Reader (Wyatt Technology Corporation, Santa Barbara, Calif.). FIG. 2 illustrates several wells of the same multiwell plate. Note that the shape of the meniscus 1, i.e., liquid/air interface, shown in FIG. 2 may vary from well to well. This variation can result in non-uniform angles of refraction and reflection from well to well of which can result in significant changes in the intensity of backscatter and secondary scatter observed by the detector. It is an objective of the invention to control the shape of the interface through which the beam departs the sample.
Evaporation can alter the sample state, skew results through altered background intensity, or prohibit light scattering measurement entirely. Partial evaporation of the solvent from a well increases the concentration of the dissolved solute which may have deleterious effects on the sample itself. Evaporation can also impact the meniscus curvature as discussed above as well as meniscus height in the well. More substantial evaporation of the sample solvent can often completely prevent accurate measurement, which is a problem particularly prevalent in very small volume multiwell plates where even a small amount of evaporation results in a large change in the height of the fluid level. Even for the larger sample volumes contained in 96 well plates, evaporation concerns prevent often useful extended measurement times as well as measurements at elevated temperature, thus making studies of temperature dependence exceedingly difficult. It is a further objective of this invention to minimize evaporation from multiwell plates.
These limitations disclosed above seriously inhibit the accurate collection of light scattering data from samples contained within the wells of multiwell plates. Means are necessary to mitigate evaporation from liquid samples contained in the wells, to increase the uniformity of background light scatter from well to well, and to reduce the intensity of background light measured by the detector. A primary objective of the present invention is to provide means by which all of these limitations for using multiwell plates for optical measurements may be mitigated allowing for an increased quality of optical measurements of samples contained in multiwell plates that have been impossible prior to the presently disclosed invention.